The present invention is an invention concerned with remedies for ischemic disease. One typical ischemic disease, obstructive arteriosclerosis, will be described first.
Obstructive arteriosclerosis is a disease in which an arteriosclerotic (atherosclerotic) lesion results in deposition of an atheromatous substance mainly consisting of fats on the endarterium, to arouse occlusion or stenosis of a major truncal artery in the extremity, especially in the lower limb, thereby causing an ischemic disorder in its periphery. Clinical symptoms of this disease are classified as coldness or numbness, intermittent claudication, rest pain, and ulcer/necrosis. In Japan, patients with obstructive arteriosclerosis are estimated to number about 100,000 (Yusuke Tada: Biomedicine & Therapeutics, Vol. 31, 289-292; 1997). The number of patients with this disease is expected to increase because of the increase in the elderly population and the westernization of diets.
Therapies of obstructive arteriosclerosis include kinesitherapy or exercise therapy, pharmacotherapy, and revascularization; which are selected depending on symptoms or the patient's condition. Other measures, now under consideration, for avoiding a resection of a severely ischemic limb are angiogenic therapies (gene therapy, bone marrow autotransplantation, etc.) for promoting angiogenesis. These therapies are currently achieving some success in the treatment of obstructive arteriosclerosis, but the respective therapies involve the following problems.
In some mild cases, the distance of walking has increased in exercise therapy. However, the effect of this therapy is difficult to predict. Moreover, patients are not satisfied with the increase in the walking distance, if any, and 30% of them are reported to have requested revascularization (Takashi Ohta: Japan Medical Journal, Vol. 3935, 25-29, 1999). Thus, at present, this therapy is not a very effective form of treatment.
In pharmacotherapy, antiplatelet agents are mainly prescribed, but they merely prevent an aggravation of symptoms. Microcirculation improving agents and oxygen transport improving agents, which have recently been developed aggressively, are only expected to be indicated for mild cases. Nowadays, there are no radical remedies available for obstructive arteriosclerosis.
Revascularization, on the other hand, is currently the most effective therapy, which involves percutaneous angioplasty or a bypass operation depending on the condition of the patient or the location or extent of the lesion. However, these surgical operations are so extensive that they pose problems, such as surgery-associated complications or death, and a poor prognosis for a long life.
Gene therapy using angiogenic factor is aimed at correcting ischemia by developing collateral circulation channels. Examples of known angiogenic factors are vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF). In Japan, clinical studies using human HGF are under way. A method, which involves its intramuscular injection into the lower limb muscle using a plasmid carrying HGF gene, has been investigated in patients with severely ischemic limbs, and expectations are growing for its efficacy. However, this therapy is still at the experimental stage, and evaluations of its safety and efficacy have not been fully carried out. Thus, gene therapy has not become popular.
Intramuscular transplantation of autologous bone marrow cells, which has recently attracted attention, is a therapy in which bone marrow cells are transplanted into the muscle near the diseased part, whereafter they are differentiated into vascular endothelial cells to form blood vessels, thereby treating the diseased part. Bone marrow autotransplantation has no adverse effects on the immune system, and has been recognized to present differentiation of bone marrow cells into endothelial cells or increase the number of blood vessels in animal models. Although its efficacy will have to be evaluated in an increased number of patients, this therapy is expected to become a promising one, because it can treat severe cases. However, the bone marrow is taken under general anesthesia in a clinical setting, so the heavy burden imposed on the patient and medical staff in taking the bone marrow may present problems.
Recent studies have shown that hematopoietic stem cells, which can differentiate into vascular endothelial cells, are present not only in the bone marrow, but also in the peripheral blood, and they take part in angiogenesis (Qun Shi et al., Blood vol. 92, 362-367, 1998; Takayuki Asahara et al., Circulation Research vol. 85, 221-228, 1999; Mario Peichev et al., Blood vol. 95, 952-958, 2000). (The hematopoietic stem cells are called “precursor cells for endothelial cells” from the viewpoint of the function of differentiating into endothelial cells. However, these cells are originally derived from hematopoietic stem cells. Thus, the term “hematopoietic stem cells” is used herein in accordance with the concept that they are a cell population capable of becoming endothelial cells.) Hence, hematopoietic stem cells in the peripheral blood are taken and transplanted into the muscle close to the diseased part, whereby treatment of obstructive arteriosclerosis can be expected. This procedure is advantageous in that the burden imposed on the patient and medical staff at the time of taking peripheral blood stem cells is less than that during transplantation of stem cells present in the bone marrow. Normally, however, the frequency of existence of hematopoietic stem cells in the peripheral blood is extremely low. Thus, it is highly questionable whether a necessary and adequate amount of hematopoietic stem cells for the treatment of obstructive arteriosclerosis can be obtained.
Human G-CSF is a hematopoietic factor discovered as a differentiation/growth factor for progenitor cells of the granulocytic lineage. It is clinically applied as a remedy for neutropenia following bone marrow transplantation or cancer chemotherapy, because it facilitates neutrophilic hematopoiesis in vivo. In addition to this action, human G-CSF acts on hematopoietic stem cells to stimulate their proliferation and differentiation, and also acts to mobilize hematopoietic stem cells present in the bone marrow into the peripheral blood. Actually, based on the latter action, transplantation of the peripheral blood hematopoietic stem cells mobilized by human G-CSF, i.e. peripheral blood stem cell transplantation, is performed in the clinical setting, with the aim of accelerating hematopoietic recovery in cancer patients after intensive chemotherapy. This hematopoietic stem cell mobilizing action of G-CSF is far more potent than that of GM-CSF, also a hematopoietic factor for the granulocytic lineage. In terms of few side effects as well, G-CSF has superiority over GM-CSF.
HGF is a protein which is produced by various mesenchymal cells and targets many epithelial cells, neurons, endothelial cells, and some mesenchymal cells. HGF is known to have cell motility promoting activity and epithelial morphogenesis (luminal structure, etc.) inducing activity, in addition to cell proliferation promoting activity. Since HGF functions as an organ regenerating factor for promoting the regeneration of the kidney, the lung and the digestive tract, as well as the liver, in adults, it is expected to be a remedy for organ disease.